The invention relates to measurements of ion mobilities in gases under the influence of electric fields. Mass spectrometers can only ever determine the ratio of the ion mass to the charge of the ion. In the following, the term “mass of an ion” or “ion mass”, which is used for the sake of simplicity, always refers to the ratio of the mass m to the number of elementary charges z of the ion. This charge-related mass m/z has the physical dimension of a mass; it is often called “mass-to-charge ratio”, although this is dimensionally incorrect. “Ion species” shall denote those ions having the same elementary composition, the same charge and the same structure. The ion species generally comprises all the ions of an isotope group, which may well include ions of slightly different masses, but virtually the same mobilities.
Isomers of the primary structure of bioorganic molecules (structural isomers) and isomers of the secondary or tertiary structure (conformational isomers) show different geometrical forms but exactly the same mass. It is therefore impossible to differentiate between them on the basis of their mass alone. Some information as to the structure can be obtained from fragment ion spectra; however, an efficient and certain way to recognize and distinguish such isomers is to separate them according to their different ion mobilities.
Today, ion mobilities are predominantly measured via the drift velocities of the ions in long drift regions. A drift region for measuring ion mobility contains an inert gas (such as helium or nitrogen). The ions of the substance under investigation are pulled through the gas by means of an electric field produced by suitable DC potentials at ring electrodes, which line the drift region. The large number of collisions with the gas molecules produces a constant drift velocity vd for each ion species which is, in first approximation, proportional to the electric field strength E: vd=K0×E. The proportionality constant K0 is called the “ion mobility” of this ion species. The ion mobility is a function of the temperature, gas pressure, type of gas, ion charge and, in particular, the collision cross-section of the ions. At the same temperature, pressure and type of gas, isomeric ions of the same charge-related mass m/z, but different collision cross-sections have different ion mobilities. Isomers of the smallest geometric dimension possess the greatest mobility and therefore the highest drift velocity through the gas. Unfolded protein ions undergo more collisions than tightly folded proteins. Protein ions which are unfolded, or partially folded, therefore arrive at the end of the cell later than strongly folded ions of the same mass. But structural isomers, for example proteins with glycosyl, lipid or phosphoryl groups at varying sites, also have different collision cross-sections, which allows them to be distinguished by measuring their ion mobility.
In modern chemical and biological research, it has become more and more important to have knowledge about the folding structures of molecules, which often can be determined by mobility measurements of their ions. Therefore devices to measure the mobility of ions have been incorporated into mass spectrometers, in particular to combine measurement of the charge-related mass of ions with measurement of collision cross-sections. The folding structures strongly influence the mechanism of action and thus the effect of the molecules in the living organism; different foldings can signify normal or abnormal functions of biopolymers in biosystems, and hence health or disease of tissue parts of even whole organisms.
A variety of information can be obtained from ion mobility measurement. It is possible to qualitatively detect simply the existence of different conformational isomers, for example. More detailed measurements of the mobility spectra can be used to quantitatively analyze mixtures of structural isomers or conformational isomers (as part of quality control for the production of chemicals, for example). Folding patterns can be confirmed or disproved by calibrated ion mobility measurements with determination of exact mobility values and comparisons with computed collision cross-sections.
A number of academic research groups have coupled ion mobility spectrometry with mass spectrometers. A pressure range of a few hectopascals has been adopted almost universally for the mobility drift region; the drift region for higher mobility resolutions is up to four meters and more, and electric field strengths of 2,000 volts per meter and more are applied. In this pressure range, the drifting ions appear to form hardly any complexes with other substances, so the mobilities of the ion species can be measured without any interference, unlike mobility measurements at atmospheric pressure. But in the long drift regions, the ions also diffuse radially over long distances, and therefore quite large diameters have to be chosen for these drift regions.
The ions are usually introduced into the drift region in the form of temporally short ion pulses, as a result of which they initially adopt the shape of spatially small ion clouds, which are pulled through the drift region by the electric field. In the gas of the drift region, these ion clouds are subject to diffusion into the surrounding space, the diffusion being caused by collisions statistically distributed in terms of spatial directions and kinetic energies due to molecular Brownian motion. The diffusion takes place in both the forward and the backward direction, and also at right angles to the drift region. The gas in the drift region is often kept at temperatures of between about 150 and 300 degrees Celsius, but can also be greatly cooled for special experiments. The mobility resolving power Rmob (“mobility resolution” for short) is influenced predominantly by this diffusion broadening of the ion clouds, especially for long drift regions and high electric field strengths; all other influences, such as the space charge, are negligibly small. The part of the mobility resolution determined by the diffusion broadening is given by the equation
            R      d        =                            zeEL          d                          kT          ⁢                                          ⁢          ln          ⁢                                          ⁢          2                      ,where z is the number of elementary charges e, E the electric field strength, Ld the length of the drift region, k the Boltzmann constant and T the temperature of the gas in the drift region. The mobility resolution is defined as Rmob=K0/ΔK0, where ΔK0 is the width of the ion signal of the mobility K0 at half height, measured in units of the mobility. The part Rd of the mobility resolution given by the diffusion is not dependent on either the type or pressure of the gas in the drift region; the mobility K0 itself, however, does depend not only on the temperature, but also on the pressure and type of the gas.
As is known from very early work on charged particles from the end of the 19th century, this type of ion mobility measurement in a non-moving drift gas can be modified by a counter-flow of the gas in the drift region, resulting in a shortening of the drift region. In this case, arbitrarily high mobility resolutions can, in theory, be achieved for ions of a selected mobility, which are held over a long period in equilibrium between the electric force of attraction and the viscous drag in the gas; but unfortunately only in theory. For practical applications there are fundamental limits which make the method unusable because the diffusion of the ion cloud, which is in equilibrium between the electric force of attraction and the viscous drag in the gas, does not stop either radially or axially at any time. Therefore, the ion cloud drifting apart by diffusion quickly exceeds all instrument dimensions.
Compared to the numerical values for mass resolutions in mass spectrometry, the mobility resolutions which can be achieved in practice are generally very low. The first commercial ion mobility spectrometer for bioorganic ions, which is very successful on the market, has a mobility resolution of only Rmob=10 to 15. With a mobility resolution of Rmob=10, two ion species whose collision cross-sections differ by only 20 percent can be readily separated.
Only highly specialized academic working groups have, as yet, been able to achieve significantly higher mobility resolutions of between Rmob=60 and 100, in rare individual cases up to Rmob=150, with drift lengths roughly between two and six meters and field strengths between 2,000 and 4,000 volts per meter, making it possible to differentiate between ion species whose mobilities differ by only one to three percent. Ion mobility spectrometers with a resolution above Rmob=60 shall be called “high resolution” here.
Since long mobility drift regions also entail strong transverse diffusion, longer drift regions must have a large diameter in order that the ions do not arrive at the wall electrodes. A well-tried method is therefore to guide the ions back to the axis of the drift region once they have passed through part of the drift region, after about two meters, for example. This is done using so-called “ion funnels”. These consist of a large number of stacked ring electrodes, closely spaced by only a few millimeters apart, whose aperture diameters taper continuously from the diameter of the drift region, between 30 and 40 centimeters, for example, to around two to five millimeters and thus form a funnel-shaped enclosed volume. The two phases of an RF voltage, usually of several megahertz and between a few tens of volts and one hundred volts, are applied alternately to the apertured diaphragms, thus generating a pseudopotential which keeps the ions away from the funnel wall. A DC electric field is superimposed on the RF voltage by a DC voltage gradient, and this electric field pushes the ions slowly to the narrow exit of the funnel and through it. It has been found that such ion funnels do not measurably reduce the mobility resolution of a long drift region.
Ion funnels are not only used to guide the ions back to the axis of the drift region in ion mobility spectrometers; they are also used in mass spectrometers in general to capture larger ion clouds and thread them into narrow ion guides. As can be seen in the example shown in FIG. 2, such ion funnels are often found in mass spectrometers with an electrospray ion source, in which ions generated outside the vacuum system are transferred, together with a curtain gas, through inlet capillaries into the vacuum, where they are captured by ion funnels and freed of most of the curtain gas. As shown schematically in FIG. 2, some mass spectrometers even contain two such ion funnels, placed in series, in order to quickly move from a region with higher pressure of a few hectopascals at the end of the inlet capillary to a region with lower pressure of between about 10−4 and 10−2 pascal. Inside these ion funnels exists a focused flow of the curtain gas, which under circumstances can even have all the characteristics of a jet flying at the speed of sound, due to the adiabatic cooling.
It should be briefly mentioned here that such gas flows are also often found in other types of ion guide, particularly if they are close to ion sources, such as the ion guide (11) of FIG. 2. Such ion guides can simply have the form of hexapole or quadrupole rod systems, which are operated with RF voltages, for example. A series of ion-optical lenses can also form an ion guide. Ion guides can be constructed with axial electric fields to actively push the ions through, although such ion guides are rare, as yet, except for ion funnels.
It is known that the conditions for conformational changes which occur by changes of the gas temperature can also be studied in ion mobility spectrometers. If, for example, the gas temperature is continuously increased in a region where the prevailing gas density and the dwell time of the ions allow that they can largely attain the temperature of the gas, and if the mobilities are measured as a function of the temperature, it is then possible to investigate transitions from one type of folding to another. It is particularly possible to determine the energy thresholds which have to be exceeded for conformational changes. Very fast cooling of the ions from a very hot state allows the most probable conformational states to be frozen and thus measured. Slow cooling of previously hot ions can often be used to find the conformational isomer with the lowest energy level.
For many biochemical applications, particularly protein chemistry applications for determining conformational states, a mass-accurate mass spectrometer, for example a time-of-flight mass spectrometer with an integrated mobility measuring station having a mobility resolution of Rmob=30-50 would already be eminently suitable. This could separate ions with mobilities differing only by some three to six percent. Conformational changes are often accompanied by mobility changes of at least this order of magnitude. This range of Rmob=30-50 shall therefore be called “medium-resolution” in the following, while the region below Rmob=20 shall be considered to be “low-resolution”. The region with Rmob>60 has already been defined above as “high-resolution”.
It should be noted here, that mobility resolution is essential for many applications, but mobility precision might be even more essential. Mobility precision is the precision for the determination of the mobility K0. The precision characterizes how well the mobility constant for a single ion species can be reproduced. With a good mobility spectrometer of Rmob=50, the mobility constant K0 may be determined with a precision of 0.2 percent or even better.
Several arrangements of mobility spectrometers are known where the ions are mass-analyzed in a high-resolution time-of-flight mass spectrometer, in addition to the measurement of their mobility, the aim being to obtain mass spectra and mobility spectra of the ion mixtures at the same time. It is of particular interest if daughter ion spectra of ions of a selected mobility can also be acquired in order to obtain additional information on the structure of the ions.
For such combinations, current types of high-resolution ion mobility spectrometer have the disadvantage of being several meters long. Such a solution is unfavorable for commercial instruments. Even medium-resolution ion mobility spectrometers with a straight drift region are about one meter long. For the construction of small, medium- to high-resolution mobility analyzers, a solution is required which reduces the overall length without diminishing the mobility resolution.
In the publication WO 2004/109741 A2 (John Noyes, priority date Jun. 6, 2003) methods and arrangements have been proposed where ions can be introduced into a laminar gas flow, kept inside the laminar gas flow by an ion guide, and pushed over the maximum of an opposing electric field of a potential barrier. Ions which are pushed over are separated from ions which are held back by the electric field opposing the gas flow. By changing the barrier, the boundary between the ions pushed over and those held back can be varied. The publication does not give a definition of the term “laminar”; however, the disclosed system is designed to produce a laminar gas flow in a tube. The tube is located inside an RF quadrupole rod ion guide and is manufactured from a high-resistance conducting dielectric material so that the RF field can penetrate the tube wall and keep the ions in the axis of the laminar gas flow. This tube and the ion guide are the essence of the invention disclosed in the publication which is completely oriented toward the gas flow in this tube with corresponding parabolic velocity profile; see here FIGS. 8 and 9 and the accompanying descriptions, for example.
Although no methods for acquiring mobility spectra are presented in this publication, it is nowadays obvious that mobility spectra can be acquired with this arrangement. However, since the publication gives no measured values at all regarding the separation of ions at the barrier, it is not possible to infer from this publication whether, and how well, the separation of ions of different mobility would work and whether a sufficiently good mobility resolution could be achieved. A fundamental disadvantage of the method presented in this publication is that a parabolic velocity profile prevails in a laminar gas flow through a tube, so only the ions on the axis experience the maximum friction, with which they can be pushed over the barrier. The RF multipole rod system must therefore produce very good focusing of the ions on the axis of the tube in order to offset this disadvantage.
In general, electric field barriers are connected with electric potential distributions, usually with potential barriers. The maximum of the electric field component of a potential barrier in opposite direction to the flowing gas will be simply called “field maximum” or “field barrier” below. The field maximum is identical to the steepest part of the positive slope of the potential distribution of the potential barrier in the direction of the gas flow.
A publication by J. S. Page et al., “Variable low-mass filtering using an electrodynamic ion funnel”, Journal of Mass Spectrometry. 2005, 40: 1215-1222 elucidated the use of an ion funnel to suppress ions of low mass in the range up to about 500 daltons, which often form a strongly interfering background in mass spectra. The authors hold back light ions below an adjustable mass threshold at the end of the ion funnel by means of an adjustable potential barrier at a ring diaphragm and filter them out of the ion current. To explain this effect, the authors propose that essentially the gas flow in the ion funnel pushes the ions over the field barrier connected with the potential barrier as a function of their mobility, and that the mobility of the light ions here gives the impression of a mass dependence because, for light ions, the mobility is mainly inversely proportional to the mass of the ions. The authors have made no attempt to use this effect to measure the ion mobility, however, despite extensive measurements on the suppression of light ions.